ab155898
6-Phosphofructokinase
Activity Assay Kit
(Colorimetric)
Instructions for Use
For the sensitive and accurate measurement of
phosphofructokinase activity in animal tissue and
cell culture samples (adherent and suspension)
This product is for research use only and is not
intended for diagnostic use.
Version 2 Last Updated 14 October 2014
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Table of Contents
1.
Overview
3
2.
Protocol Summary
4
3.
Components and Storage
5
4.
Assay Protocol
7
5.
Data Analysis
10
6.
Troubleshooting
12
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1. Overview
Phosphofructokinase (PFK) is a key glycolytic enzyme and plays a
major regulatory role during glycolysis. This enzyme is present in
bacteria, plants and animals. There are 2 types of PFKs - PFK1 and
PFK2. In the presence of ATP, PFK1 & PFK2 catalyzes the
conversion of fructose-6-phosphate to fructose-1,6-diphosphate and
fructose-2,6-diphosphate respectively and ADP. PFK has 3 major
isoforms in mammals: PFK-M (muscle), PFK-L (liver) and PFK-P
(platelet). In humans, PFK deficiency causes glycogen storage
disease, also called Tarui’s disease, which is characterized by
exercise-induced muscle weakness and cramps. On the other hand,
increased PFK activity contributes to cancer cell proliferation and
tumorigenicity. Early detection of abnormal phosphofructokinase
activity is crucial for diagnosis, prediction and therapeutic strategy.
In Abcam's 6-Phosphofructokinase Activity Assay kit (ab155898),
PFK
converts
fructose-6-phosphate
and
ATP
to
fructose-
diphosphate and ADP. The ADP in the presence of substrate and
enzyme mix is converted to AMP and NADH, which reduces a
colorless probe to a colored product with strong absorbance at 450
nm. PFK activity assay is simple, robust, and rapid and can detect
phosphofructokinase activity less than 1 mU.
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2. Protocol Summary
Reagent Preparation
Standard Curve Preparation
Sample Preparation
Positive Control
Reaction Mix
Measurement and Calculation
4
3. Components and Storage
A. Kit Components
Item
Quantity
Assay Buffer
27 mL
PTK Substrate (Lyophilized)
1 vial
ATP (Lyophilized)
1 vial
Enzyme Mix (Lyophilized)
1 vial
Developer (Lyophilized)
1 vial
NADH Standard (Lyophilized)
1 vial
Positive Control (Lyophilized)
1 vial
* Store the kit at-20°C and protect from light. Please read the entire
protocol before performing the assay. Avoid repeated freeze/thaw
cycles. Warm all Buffers to room temperature before use. Briefly
centrifuge all small vials prior to opening.
B. Additional Materials Required

96-well clear plate with flat bottoms

Multi-well spectrophotometer (ELISA reader)
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4. Assay Protocol
A. Reagent Preparation
1.
PFK Substrate:
Reconstitute with 220 μl Assay Buffer. Store at -20°C. Use within
two months. Keep on ice while in use.
2.
ATP:
Reconstitute with 220 μl dH2O. Store at -20°C. Use within two
months. Keep on ice while in use.
3. Enzyme Mix:
Reconstitute with 220 μl Assay Buffer. Pipette up and down to
dissolve completely. Aliquot and store at -20°C. Avoid repeated
freeze/thaw cycles. Use within two months. Keep on ice while in
use.
4. Developer:
Reconstitute with 220 μl dH2O. Pipette up and down to dissolve
completely. Store at -20°C. Use within two months.
5. NADH Standard:
Reconstitute with 40 μl Assay Buffer to generate 10 mM NADH
Standard stock solution. Store at –20°C. Use within two months.
Keep on ice while in use.
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6. Positive Control:
Reconstitute with 100 μl Assay Buffer and mix thoroughly.
Aliquot and store at –20°C.
B. Phosphofructokinase Activity Assay Protocol
1. NADH Standard Curve:
Dilute NADH Standard 10 fold by adding 10 μl NADH Standard
stock solution to 90 μl Assay Buffer. Add 0, 2, 4, 6, 8 and 10 μl of
1 mM diluted NADH Standard into a series of wells in 96 well
plate to generate 0, 2, 4, 6, 8 and 10 nmol/well of NADH
Standard. Adjust final volume to 50 μl/well with Assay Buffer.
2. Sample Preparation:
Rapidly homogenize tissue (20 mg) or cells (2 x 106) with 200 μl
ice cold Assay Buffer for 10 minutes on ice. Centrifuge at 12000
rpm for 5 min. Collect the supernatant. Add 1-50 μl sample
(100 μg) per well, adjust final volume to 50 μl with Assay Buffer.
Prepare a parallel sample well as the background control to
avoid interference from ADP and NADH in the sample.
Note: For unknown samples, we suggest testing several doses
to ensure the readings are within the standard curve range.
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3. Positive Control:
Add 10-20 μl of diluted PFK Positive Control into well(s). Adjust final
volume to 50 μl with Assay Buffer.
4. Reaction Mix:
Mix enough reagents for the number of assays to be performed.
For each well, prepare 50 μl Reaction Mix containing:
Reaction Mix
Background
Control Mix
Assay Buffer
42 µl
44 µl
Enzyme Mix
2 µl
2 µl
Developer
2 µl
2 µl
ATP
2 µl
2 µl
PTK Substrate
2 µl
---
Add 50 μl of the Reaction Mix to each well containing the
Standard, Positive Control and test samples and 50 μl of
Background Control mix to each well containing the Background
Control sample. Mix well.
5. Measurement:
Incubate for 20-60 min at 370C and measure OD450nm.
Note: Incubation time depends on the Phosphofructokinase
activity in the samples. We recommend measuring the OD in a
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kinetic mode, and choose two time points (T1 & T2) in the linear
range to calculate the PFK activity of the samples. The NADH
standard curve can read in Endpoint mode (i.e., at the end of
incubation time).
5. Data Analysis
Calculation: Subtract the 0 standard reading from all standard
readings.
Plot
the
NADH
standard
curve.
Correct
sample
background by subtracting the value derived from the background
control from all sample readings. Calculate the hexokinase activity of
the test sample: ΔOD = A2 – A1. Apply the ΔOD to the NADH
standard
curve
to
get
B
nmol
of
NADH
generated
by
Phosphofructokinase during the reaction time (ΔT = T2 - T1).
Sample
Phosphofructokinase =
Activity
B
(ΔT x V)
x
Dilution
Factor
= nmol/min/ml/ = mU/ml
Where:
B is the NADH amount from standard curve (nmol).
ΔT is the reaction time (min).
V is the sample volume added into the reaction well (ml).
Unit Definition: One unit of phosphofructokinase is the amount of
enzyme that will generate 1.0 μmol of NADH per min at at pH 7.4 at
370C.
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Figure 1: NADH standard curve (a). Phosphofructokinase activity in
bovine liver and HeLa cell lysate (b). Assays were performed
following kit protocol.
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6. Troubleshooting
Problem
Reason
Solution
Assay not
working
Assay buffer at
wrong temperature
Assay buffer must not be chilled
- needs to be at RT
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Unexpected
results
Samples
with
inconsistent
readings
Re-read and follow the protocol
exactly
Ensure you are using
appropriate reader and filter
settings (refer to datasheet)
Fluorescence: Black plates
(clear bottoms);
Luminescence: White plates;
Colorimetry: Clear plates.
If critical, datasheet will indicate
whether to use flat- or U-shaped
wells
Measured at wrong
wavelength
Use appropriate reader and filter
settings described in datasheet
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
Troubleshoot and also consider
deproteinizing samples
Use recommended samples
types as listed on the datasheet
Concentrate/ dilute samples to
be in linear range
Unsuitable sample
type
Refer to datasheet for details
about incompatible samples
Samples prepared in
the wrong buffer
Use the assay buffer provided
(or refer to datasheet for
instructions)
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Samples not
deproteinized (if
indicated on
datasheet)
Cell/ tissue samples
not sufficiently
homogenized
Too many freezethaw cycles
Samples contain
impeding substances
Samples are too old
or incorrectly stored
Lower/
Higher
readings in
samples
and
standards
Not fully thawed kit
components
Out-of-date kit or
incorrectly stored
reagents
Reagents sitting for
extended periods on
ice
Incorrect incubation
time/ temperature
Incorrect amounts
used
Standard
curve is not
linear
Not fully thawed kit
components
Pipetting errors when
setting up the
standard curve
Use the 10kDa spin column
(ab93349)
Increase sonication time/
number of strokes with the
Dounce homogenizer
Aliquot samples to reduce the
number of freeze-thaw cycles
Troubleshoot and also consider
deproteinizing samples
Use freshly made samples and
store at recommended
temperature until use
Wait for components to thaw
completely and gently mix prior
use
Always check expiry date and
store kit components as
recommended on the datasheet
Try to prepare a fresh reaction
mix prior to each use
Refer to datasheet for
recommended incubation time
and/ or temperature
Check pipette is calibrated
correctly (always use smallest
volume pipette that can pipette
entire volume)
Wait for components to thaw
completely and gently mix prior
use
Try not to pipette too small
volumes
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Incorrect pipetting
when preparing the
reaction mix
Air bubbles in wells
Concentration of
standard stock
incorrect
Errors in standard
curve calculations
Use of other
reagents than those
provided with the kit
Always prepare a master mix
Air bubbles will interfere with
readings; try to avoid producing
air bubbles and always remove
bubbles prior to reading plates
Recheck datasheet for
recommended concentrations of
standard stocks
Refer to datasheet and re-check
the calculations
Use fresh components from the
same kit
13
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